Over-expression of a cacao class I chitinase gene in Theobroma cacao L. enhances resistance against the pathogen, Colletotrichum gloeosporioides

Theobroma cacao L. plants over-expressing a cacao class I chitinase gene (TcChi1) under the control of a modified CaMV-35S promoter were obtained by Agrobacterium-mediated transformation of somatic embryo cotyledons. Southern blot analysis confirmed insertion of the transgene in eight independent lines. High levels of TcChi1 transgene expression in the transgenic lines were confirmed by northern blot analysis. Chitinase activity levels were measured using an in vitro fluorometric assay. The transgene was expressed at varying levels in the different transgenic lines with up to a sixfold increase of endochitinase activity compared to non-transgenic and transgenic control plants. The in vivo antifungal activity of the transgene against the foliar pathogen Colletotrichum gloeosporioides was evaluated using a cacao leaf disk bioassay. The assay demonstrated that the TcChi1 transgenic cacao leaves significantly inhibited the growth of the fungus and the development of leaf necrosis compared to controls when leaves were wound inoculated with 5,000 spores. These results demonstrate for the first time the utility of the cacao transformation system as a tool for gene functional analysis and the potential utility of the cacao chitinase gene for increasing fungal pathogen resistance in cacao.     

Endophytic Colonisation of Opium Poppy, Papaver somniferum, by an Entomopathogenic Beauveria bassiana Strain

Beauveria bassiana strain EABb 04/01-Tip isolated from stem-borer larvae of Timaspis papaveris (Hymenoptera: Cynipidae), a serious pest of opium poppy in Spain, was shown to be able to become established endophytically in this pharmaceutical crop. Microbiological, molecular and light and electron microscopic methods were used to study fungal colonisation and to describe its mode of penetration. After inoculation with a foliar spray of conidia, microbiological methods showed 100% of plants examined 24, 48, 72 and 144 h after treatment to be colonised endophytically by the fungus, although the percentage of previously surface sterilised leaf pieces showing fungal growth was 100% at 24 and 48 h, and 80 and 75% at 72 and 144 h after treatment, respectively. The fungus was also observed in leaf pieces obtained from newly formed leaves, indicating that it could spread from treated leaves to leaves formed after fungal application. For molecular studies, a polymerase chain reaction (PCR) protocol was used to amplify the ITS1-5.8S-ITS2 regions of the rDNA of the plant and the fungus. This procedure allowed the detection of the fungus on the surface of the leaves and also endophytically, but only at 72 h after treatment. A nucleotide BLAST search revealed that the ITS1-5.8S-ITS2 sequence of strain EABb 04/01-Tip showed 100% homology with a similar sequence from Cordyceps bassiana. SEM images revealed that although numerous conidia were observed on the leaf surface, few germinated and penetrated. Intracellular colonisation by B. bassiana was not observed, but hyphae were detected growing into the xylem vessels. The fungus was found to colonise 40.5 ± 4.3% of seedlings (with two cotyledons and the two first real leaves) from seeds dressed with a fungal spore suspension. These results may have implications in the biological control of T. papaveris, including the possible systemic protection of the plant against this cynipid.     

Isolation of endophytic endospore-forming bacteria from Theobroma cacao as potential biological control agents of cacao diseases

Sixty-nine endospore-forming bacterial endophytes consisting of 15 different species from five genera were isolated from leaves, pods, branches, and flower cushions of Theobroma cacao as potential biological control agents. Sixteen isolates had in vitro chitinase production. In antagonism studies against cacao pathogens, 42% inhibited Moniliophthora roreri, 33% inhibited Moniliophthora perniciosa, and 49% inhibited Phytophthora capsici. Twenty-five percent of isolates inhibited the growth of both Moniliophthora spp., while 22% of isolates inhibited the growth of all three pathogens. Isolates that were chitinolytic and tested negative on Bacillus cereus agar were tested with in planta studies. All 14 isolates colonized the phyllosphere and internal leaf tissue when introduced with Silwet L-77, regardless of the tissue of origin of the isolate. Eight isolates significantly inhibited P. capsici lesion formation (p = 0.05) in detached leaf assays when compared to untreated control leaves. ARISA with bacilli specific primers amplified 21 OTUs in field grown cacao leaves, while eubacteria specific primers amplified 58 OTUs. ARISA analysis of treated leaves demonstrated that inundative application of a single bacterial species did not cause a long-term shift of native bacterial communities. This research illustrates the presence of endospore-forming bacterial endophytes in cacao trees, their potential as antagonists of cacao pathogens, and that cacao harbors a range of bacterial endophytes.     

Flooding effects on rapid responses of the invasive plant Alternanthera philoxeroides to defoliation

In experiments under controlled growth conditions it was examined how flooding affected the responses of the invasive plant Alternanthera philoxeroides to defoliation. In drained and flooded conditions, plants were subjected to five defoliation levels: 0, 10, 50, 90% removal of leaf tissue and apex removal (90% leaf tissue plus apical bud removal). Plants were harvested weekly for five weeks. In drained conditions, plant biomasses including total biomass, shoot biomass and root biomass after 50% defoliation rapidly recovered to the control plant level. They were significantly lower for the 90% defoliation and apex removal treatments compared to control plants throughout the experiment. In flooded conditions, total biomass and shoot biomass after 50% defoliation, 90% defoliation, and apex removal treatments could return to control plant levels before the end of the experiment. In 90% defoliation and apex removal treatments root to shoot biomass ratios of both drained and flooded plants were initially much higher than in control plants, but the difference disappeared rapidly. The final biomasses decreased with increased defoliation intensity in drained conditions, but no significant difference was generally found in any of the defoliation treatments in flooded conditions. The rapid re-growth of A. philoxeroides plants after defoliation may partly be responsible for its invasion success. However, defoliation capable of removing 90% of the leaf tissue may be desirable in restricting the growth of this invasive species in drained conditions.     

The influence of emergent trees on rainfall distribution in a cacao agroforest (Sulawesi,Indonesia)

Emergent trees may have an influence on the volume and the spatial distribution of water input into agroforestry stands and may thus affect water availability for the main crops. Our goal was to analyze the influence of such trees on rainfall distribution in a cacao agroforest area in the rainforest margin zone of Central Sulawesi, Indonesia. The emergent trees studied belong to the species Bischofia javanica (Phyllanthaceae) and were 15 m high remnants from the natural forest. A set of 96 throughfall gauges was systematically distributed underneath canopies of cacao only, and underneath canopies of cacao plus emergent trees (cacao plus trees). From an earlier study we knew that stemflow can safely be estimated with less than 1% of gross precipitation (Pg).     

Partitioning of carbon and nitrogen and the nutrition of root and shoot apex in a nodulated legume

Empirically based models depicting exchanges of C, N, and H₂O in phloem and xylem among organs of nodulated white lupin (Lupinus albus cv Ultra) were constructed for the interval 51 to 58 days after sowing. Information was incorporated on the economy of C, N, and H₂O in plant parts, the solute composition of transport fluids collected at selected sites on the plant, and the photosynthetic inputs, transpirational losses, and translocatory activities of different age groups of leaflets and stem + petiole segments of the shoot. Partitioning of C and N showed preferential transfer of N to the shoot apex, which imported 13 milligrams C per milligram N, compared with 54 milligrams C per milligram N for the nodulated root. Leaves translocated assimilates at a C:N weight ratio of 43 to 59, and older leaves serving the roots produced the translocate most rich in N relative to C. The shoot apex was enriched with N, additional to its intake from leaves, by direct uptake of xylem fluid (C:N ratio, 2.4) and receipt of nitrogenous solutes transferred from xylem to upward-moving phloem streams in upper regions of the stem. The models for flow of N and H₂O indicated that xylem streams passing to leaves were substantially less rich in N than the adjacent stream moving through the body of the stem and that a progressive increase in concentration of N occurred within stem xylem elements from base to top of the shoot. This apparently resulted from an abstraction of N from xylem of departing leaf traces, possibly by xylem transfer cells, and a subsequent feedback of this N to xylem streams passing on up the shoot. Upper leaves and shoot apex, therefore, acquired more N from xylem per unit of H₂O transpired than lower parts of the shoot.     

Salicylic acid and salicylic acid glucoside in xylem sap of <Emphasis Type="Italic">Brassica napus</Emphasis> infected with <Emphasis Type="Italic">Verticillium longisporum</Emphasis>

Salicylic acid (SA) and its glucoside (SAG) were detected in xylem sap of Brassica napus by HPLC–MS. Concentrations of SA and SAG in xylem sap from the root and hypocotyl of the plant, and in extracts of shoots above the hypocotyl, increased after infection with the vascular pathogen Verticillium longisporum. Both concentrations were correlated with disease severity assessed as the reduction in shoot length. Furthermore, SAG levels in shoot extracts were correlated with the amount of V. longisporum DNA in the hypocotyls. Although the concentration of SAG (but not SA) in xylem sap of infected plants gradually declined from 14 to 35 days post infection, SAG levels remained significantly higher than in uninfected plants during the whole experiment. Jasmonic acid (JA) and abscisic acid (ABA) levels in xylem sap were not affected by infection with V. longisporum. SA and SAG extend the list of phytohormones potentially transported from root to shoot with the transpiration stream. The physiological relevance of this transport and its contribution to the distribution of SA in plants remain to be elucidated.     

盐地碱蓬二型性种子及其幼苗对盐渍环境的适应性

研究了盐地碱蓬二型性种子中离子含量与刚萌发幼苗耐盐性之间的关系,以及盐分对砂培盐地碱蓬二型性种子的幼苗生长、离子含量及光合特性的影响.棕色种子中离子含量显著高于黑色种子.与对照相比,100和400 mmol/L NaCl对棕色种子幼苗伸长没有抑制作用,却显著抑制黑色种子幼苗的伸长.NaCl处理下棕色种子的幼苗地上部分干重和主茎一级分枝数比黑色种子幼苗高,但二型性种子的幼苗叶片中离子含量、叶绿素含量及光合放氧速率却没有明显差异.上述结果说明盐地碱蓬棕色种子较高的离子含量可能是棕色种子刚萌发幼苗耐盐性较强的重要原因.棕色种子幼苗较高的生物量可能与其较多的分枝数有关.二型种子的这些特征可能决定了其在群落建成中所起到的不同作用.     

Mistletoes and mutant albino shoots on woody plants as mineral nutrient traps

Background and Aims

Potassium, sulphur and zinc contents of mistletoe leaves are generally higher than in their hosts. This is attributed to the fact that chemical elements which are cycled between xylem and phloem in the process of phloem loading of sugars are trapped in the mistletoe, because these parasites do not feed their hosts. Here it is hypothesized that mutant albino shoots on otherwise green plants should behave similarly, because they lack photosynthesis and thus cannot recycle elements involved in sugar loading.

Methods

The mineral nutrition of the mistletoe Scurrula elata was compared with that of albino shoots on Citrus sinensis and Nerium oleander. The potential for selective nutrient uptake by the mistletoe was studied by comparing element contents of host leaves on infected and uninfected branches and by manipulation of the haustorium–shoot ratio in mistletoes. Phloem anatomy of albino leaves was compared with that of green leaves.

Key Results

Both mistletoes and albino leaves had higher contents of potassium, sulphur and zinc than hosts or green leaves, respectively. Hypothetical discrimination of nutrient elements during the uptake by the haustorium is not supported by our data. Anatomical studies of albino leaves showed characteristics of release phloem.

Conclusions

Both albino shoots and mistletoes are traps for elements normally recycled between xylem and phloem, because retranslocation of phloem mobile elements into the mother plant or the host is low or absent. It can be assumed that the lack of photosynthetic activity in albino shoots and thus of sugars needed in phloem loading is responsible for the accumulation of elements. The absence of phloem loading is reflected in phloem anatomy of these abnormal shoots. In mistletoes the evolution of a parasitic lifestyle has obviously eliminated substantial feeding of the host with photosynthates produced by the mistletoe.     

Transport, synthesis and catabolism of abscisic acid (ABA) in intact plants of castor bean (Ricinus communis L.) under phosphate deficiency and moderate salinity

Flows of abscisic acid (ABA) were investigated in whole plantsof castor bean (Ricinus communis) grown in sand culture undereither phosphate deficiency or moderate salinity. Xylem transportof ABA in P-deficient plants was stimulated by a factor of 6whereas phloem transport was affected only very slightly. ABAdeposition into leaves of P-deficient plants was not appreciablydifferent from the controls because of strong net degradationin leaves. Since conjugation of ABA was strongly reduced inall organs of P-deficient plants ABA was presumably metabolizedmainly to phaseic acid and dihydrophaseic acid. The increasedimport of ABA occurred predominantly into fully differentiatedbut not senescent leaves and showed a good correlation withthe inhibition of leaf conductance under P deficiency. As with low-P-plants salt stress increased ABA synthesis inroots and associated transport in the xylem. However, salinitycaused a distinctly greater accumulation of ABA in the leaves,stem segments and the apex than in P-deficient plants. As opposedto P deficiency, ABA export in the phloem from the leaves wasstimulated by salinity. Modelling of ABA flows within an individualleaf over its life cycle showed that young growing leaves importedABA from both phloem and xylem, whereas the adult non-senescentleaves were a source of ABA and thus provided a potential shoot-to-rootstress signal as well as an acceptor for reciprocal signalsfrom root to shoot. In senescing leaves ABA flows and accumulationwere somewhat retarded and ABA was lost in net terms by exportfrom the leaf. Key words: Abscisic acid, phosphorus deficiency, salt stress, phloem and xylem transport     

The Relationship Between Size of Shoot Apices and Morphological Features of Mature Leaves and Stems of Four Species of Angiosperm Trees

Shoot apex, leaf and stem growth parameters for four speciesof deciduous trees were measured. Only in elm was there a correlationbetween the size of shoot apical meristems and mature leaves.In ash, basswood and cottonwood there was no significant differencebetween size of shoot apices of sucker and canopy branches,despite significant differences in lamina size. In the suckerbranches of all species studied there occurred an early, lateralexpansion of the subapical region of the shoot apical meristem.This correlated well with the greater diameter of stem and pithregions of sucker branches. In addition, the season's annualring of xylem was greater in basswood, cottonwood and elm. Diametersof vessel elements were greater in sucker than canopy branchesin three of the four species. Total branch and internode andnumber of nodes per branch were significantly greater for suckerbranches than canopy growth of all species studied. A hypothesis is proposed to explain the development of the verylarge surface area of leaves on sucker branches. This hypothesisis based on the position of sucker branches in relation to theroot system and involves differences in water stress known tobe present in all plants.Copyright 1993, 1999 Academic Press Sucker leaves, canopy leaves, Fraxìnus pennsylvanica Marsh, green ash, Ulmus amerìcana L., American elm, Populus deltoides Marsh, cottonwood, Tilia americana, basswood     

Neck rot (Botrytis allii) of bulb onions

Experiments on neck rot of onions, caused by Botrytis allii showed that, although the disease only became evident in store, a major source of the pathogen was samples of infected seeds. In 1972 and 1973, 39·5 and 71·4% respectively of commercial onion seed samples tested at Wellesbourne were infected. The pathogen was internal in seed and persisted for 3 ½ yr in infected seeds kept in a seed store at 10°C and 50% r.h. Seedlings raised from diseased seeds became infected by mycelial invasion of the cotyledon leaf tips from seed-coats many of which remained attached to the cotyledons when seedlings emerged from the soil. The fungus attacked the living tissues of these leaves symptomlessly, producing conidiophores only after the leaf tissue senesced and became necrotic. Because the fungus was symptomless, the rate of spread of the pathogen in onion crops was assessed by incubating successive samples of plants from the field in humid conditions when infected tissues developed conidiophores of the fungus. This method showed that the disease was progressive in onion crops spreading more rapidly in wet humid conditions (e.g. 1972) than in dry ones (e.g. 1973). The principal means of spread were probably fungal spores; conidiophores bearing spores being produced abundantly on plants in the field under high humidity. The fungus invaded the leaves of plants successively, first infecting each leaf at the tip and then growing downwards in the tissues and eventually invading the neck of the onion bulb via the leaves which emerged directly from the top of the neck. By harvest, the fungus was situated deep within the neck tissues of infected maturing onion bulbs.     

Trichoderma theobromicola and T. paucisporum: two new species isolated from cacao in South America

Trichoderma theobromicola and T. paucisporum spp. nov. are described. Trichoderma theobromicola was isolated as an endophyte from the trunk of a healthy cacao tree (Theobroma cacao, Malvaceae) in Amazonian Peru; it sporulates profusely on common mycological media. Trichoderma paucisporum is represented by two cultures that were obtained in Ecuador from cacao pods partially infected with frosty pod rot, Moniliophthora roreri; it sporulates sporadically and most cultures remain sterile on common media and autoclaved rice. It sporulates more reliably on synthetic low-nutrient agar (SNA) but produces few conidia. Trichoderma theobromicola was reintroduced into cacao seedlings through shoot inoculation and was recovered from stems but not from leaves, indicating that it is an endophytic species. Both produced a volatile/diffusable antibiotic that inhibited development of M. roreri in vitro and on-pod trials. Neither species demonstrated significant direct in vitro mycoparasitic activity against M. roreri.     

Isolation of an alpha-methylene-gamma-butyrolactone derivative, a toxin from the plant pathogen Lasiodiplodia theobromae

Lasiodiplodia theobromae is known as a multi-infectious microorganism that causes considerable crop damage, particularly to tropical fruits. When the fruits are infected by L. theobromae, the typical symptom is the appearance of black spots on the surface of the infected fruit. When injected in to the peel of banana, the culture filtrate of L. theobromae induced formation of black spots. The structure of the isolated compound responsible for this effect was determined to be (3S,4R)-3-carboxy-2-methylene-heptan-4-olide on the basis of analysis of MS, IR, and 1H and 13C NMR spectroscopic data, including HMQC, HMBC, and 1H-1H COSY experiments. The active compound was not only isolated from the culture filtrate derived from potato dextrose medium, but also from the extract of infected peels of bananas.     

The influence of supra-optimal root-zone temperatures on growth and stomatal conductance in Capsicum annuum L

Pepper (Capsicum annuum L.) plants were grown aeroponically in a Singapore greenhouse under natural diurnally fluctuating ambient shoot temperatures, but at two different root-zone temperatures (RZTs): a constant 20 +/- 2 degrees C RZT and a diurnally fluctuating ambient (A) (25-40 degrees C) RZT. Plants grown at 20-RZT had more leaves, greater leaf area and dry weight than A-RZT plants. Reciprocal transfer experiments were conducted between RZTs to investigate the effect on plant growth, stomatal conductance (gs) and water relations. Transfer of plants from A-RZT to 20-RZT increased plant dry weight, leaf area, number of leaves, shoot water potential (psi shoot), and gs; while transfer of plants from 20-RZT to A-RZT decreased these parameters. Root hydraulic conductivity was measured in the latter transfer and decreased by 80% after 23 d at A-RZT. Transfer of plants from 20-RZT to A-RZT had no effect on xylem ABA concentration or xylem nitrate concentration, but reduced xylem sap pH by 0.2 units. At both RZTs, gs measured in the youngest fully expanded leaves increased with plant development. In plants with the same number of leaves, A-RZT plants had a higher gs than 20-RZT plants, but only under high atmospheric vapour pressure deficit. The roles of chemical signals and hydraulic factors in controlling gs of aeroponically grown Capsicum plants at different RZTs are discussed.     

三种红树林植物健康与非健康叶片中真菌的比较

红树林叶片感染真菌后可能会向非健康状态变化,为了比较三种红树林植物海漆、秋茄和桐花树健康与非健康叶片真菌类群,明确其中真菌类群的差异,该研究从广西茅尾海红树林自然保护区采集海漆、秋茄和桐花树健康与非健康叶片,对叶片中真菌分离纯化并进行形态学鉴定,提取真菌的DNA,采用RAPD多样性、ITS序列对真菌进行分子鉴定。经过初步分析,从海漆、秋茄和桐花树中共分离到157株真菌,经过形态学和RAPD分析,可能为19种不同的真菌。采用真菌ITS序列对19种真菌代表性菌株进行分析,结果表明:19种真菌都属于子囊菌门,有15株与已有的真菌ITS序列相似性在97%以上;有4株相似性低于95%,可能为新种。海漆健康和非健康叶片分别分离到真菌7种和5种,健康叶片含了非健康叶片中所有真菌;秋茄健康和非健康叶片分离到真菌1种和9种,且健康叶片中分离到的内生真菌Dothiorella aegiceri也包含于非健康叶片分离的真菌中;桐花树健康和非健康叶片分离到真菌1种和3种,且桐花树分离的内生真菌也是Dothiorella aegiceri,其包含于非健康叶片中分离到的真菌。该研究证明,海漆内生真菌同时也可能是植物病原菌,内生真菌与植物病原菌没有明显界限;另一方面,该研究发现秋茄和桐花树叶片的病变是由外部的病原真菌感染引起,感染的原因可能是昆虫叮咬红树林植物后留下了创口,这些创口有利于真菌的感染。     

Polyphenoloxidase activity in coffee leaves and its role in resistance against the coffee leaf miner and coffee leaf rust

In plants, PPO has been related to defense mechanism against pathogens and insects and this role was investigated in coffee trees regarding resistance against a leaf miner and coffee leaf rust disease. PPO activity was evaluated in different genotypes and in relation to methyl-jasmonate (Meja) treatment and mechanical damage. Evaluations were also performed using compatible and incompatible interactions of coffee with the fungus Hemileia vastatrix (causal agent of the leaf orange rust disease) and the insect Leucoptera coffeella (coffee leaf miner). The constitutive level of PPO activity observed for the 15 genotypes ranged from 3.8 to 88 units of activity/mg protein. However, no direct relationship was found with resistance of coffee to the fungus or insect. Chlorogenic acid (5-caffeoylquinic acid), the best substrate for coffee leaf PPO, was not related to resistance, suggesting that oxidation of other phenolics by PPO might play a role, as indicated by HPLC profiles. Mechanical damage, Meja treatment, H. vastatrix fungus inoculation and L. coffeella infestation caused different responses in PPO activity. These results suggest that coffee resistance may be related to the oxidative potential of the tissue regarding the phenolic composition rather than simply to a higher PPO activity.